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Antimicrobial Agents and Chemotherapy, March 2001, p. 996-997, Vol. 45, No. 3
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.3.996-997.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
LETTERS TO THE EDITOR
Two Structurally Distinct VanA Resistance Elements at Different
Locations in a Glycopeptide-Resistant Strain of Enterococcus
faecalis
 |
LETTER |
Glycopeptide-resistant enterococci (GRE) can carry more than one
VanA element (3, 5), but we are unaware of previous evidence for separate transfer of such elements. Enterococcus faecalis JS3B was examined as one of 46 GRE isolated from fecal screens of a hematology patient at Addenbrooke's Hospital, Cambridge, United Kingdom (7). Overlapping PCR (1)
showed that this strain contained a group H element (11).
VanA resistance appeared to be associated with a 35-MDa plasmid, but
Palepou et al. (6) suggested that group H elements reside
on the chromosome. Strain JS3B was therefore investigated for carriage
of multiple VanA elements.
Transfer of vancomycin resistance was performed by cross-streak
conjugation (10). The recipient strains were E. faecalis JH2-2 and E. faecium GE-1, which are resistant
to fusidic acid and rifampin and lack pheromone response genes
prgA and prgB (see below). After incubation at
37°C for 72 h, the transconjugants were selected on brain heart
infusion agar (Oxoid) containing 100 µg of rifampin per ml, 25 µg
of fusidic acid per ml, and 10 µg of vancomycin per ml. Plates were
incubated at 37°C and examined daily for 5 days. Eight to 12 individual colonies were subcultured onto Columbia horse blood agar.
The colonial characteristics of selected transconjugants were noted,
and plasmid profiles were examined following alkaline lysis and agarose
gel electrophoresis (8).
Five transconjugants, representing each combination of plasmid profile,
hemolysis reaction, and colonial morphology, were subjected to
overlapping PCR (1, 11). Plasmids were resubjected to
alkaline lysis, followed by Southern blotting onto a nylon membrane,
and hybridized with a digoxigenin-labeled vanA-specific probe (2). Strain JS3B and the five transconjugants were
also examined for the conserved pheromone response genes
prgA (which encodes entry exclusion protein) and
prgB (which encodes aggregation substance protein) with
previously described PCR primers (4) and the same cycling
conditions as for overlapping PCR.
Three transconjugants represented the E. faecalis JH2-2 host
and contained group U elements. Two transconjugants represented E. faecium GE-1; one contained a group U element, whereas
the other had a group H element (Table
1). Each of the four transconjugants with
group U elements possessed a plasmid of ca. 35 MDa that hybridized with
the vanA probe. These four transconjugants and JS3B all
possessed prgA and prgB. The transconjugant with
the group H element did not possess detectable plasmids, and the
vanA probe hybridized only with residual chromosomal DNA.
However, it is possible that the group H element may be carried on a
large conjugative plasmid not recovered by the alkaline lysis technique
employed here (9). The transconjugant with the group H
element lacked pheromone response genes prgA or
prgB, suggesting that transfer of its VanA resistance element did not depend on a pheromone-responsive conjugative plasmid.
In conclusion, strain JS3B contained two distinct VanA elements, a
group U element carried on a pheromone-responsive plasmid of ca. 35 MDa
and a group H element that appeared to be chromosomal. In overlapping
PCR, the group H element, which yielded multiple amplicons, masked the
group U element, which yielded fewer amplicons (11). These
results stress the importance of using multiple molecular techniques to
identify the structure and location of VanA elements in GRE.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the PHLS Small Scientific
Initiative Fund.
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FOOTNOTES |
*
Phone:
(44)-1223-257035 Fax: (44)-1223-242775
| |
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Catherine H. Tremlett*
Derek F. J. Brown
Clinical Microbiology and Public Health Laboratory
Addenbrooke's Hospital
Cambridge CB2 2QW
United Kingdom
|
| | | | |
Marie-France I. Palepou
Neil Woodford
Antibiotic Resistance Monitoring and
Reference
Laboratory
Central Health Public Laboratory
London
NW9 5HT
United Kingdom
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Antimicrobial Agents and Chemotherapy, March 2001, p. 996-997, Vol. 45, No. 3
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.3.996-997.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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